藻胆蛋白在大肠杆菌中的重组表达及其光谱学性质与抗氧化活性研究

Optimization of Expression of Recombinant Phycobiliproteins in Escherichia coli and Assessment of Their Spectral Properties and Antioxidant Activity

摘要:

在大肠杆菌中进行了藻胆蛋白的重组表达，对重组藻胆蛋白进行了分离纯化，测定了重组藻胆蛋白的光谱学性质和抗氧化活性。结果表明，以IPTG为诱导物时，当菌液OD600为0.95时，加入终浓度为0.1 mmol/L的IPTG和5.0 mmol/L的5-氨基酮戊酸（ALA），在18 ℃条件下诱导24 h重组蛋白可获得较好表达效果，表达量达149.5 mg/L；以乳糖为诱导物时，当菌液OD600为1.2时，加入终浓度为1.0 g/L的乳糖和5 mmol/L的ALA，在18 ℃条件下诱导26 h重组蛋白可获得较好表达效果，表达量达102.9 mg/L。重组藻胆蛋白最大吸收峰位于550 nm，最大荧光发射峰位于562 nm，相对于IPTG诱导表达的重组藻胆蛋白，经乳糖诱导表达重组藻胆蛋白，其550 nm处具有较强的光吸收，562 nm处具有较高荧光强度，同时也具有较高的羟基自由基和超氧阴离子的清除能力。

Abstract:

Recombinant phycobiliproteins were expressed in Escherichia coli. The overexpressed phycobiliproteins were isolated and purified, and their spectral properties and antioxidant activities were determined. The optimal conditions for the induction of recombinant phycobiliproteins with isopropyl-beta-D-thiogalactopyranoside (IPTG) were identified as following: adding 0.1 mmol/L of IPTG and 5 mmol/L of 5-aminolevulinic acid (5-ALA) into the growth medium at an optical density at 600 nm (OD600) of 0.95 and then incubating the cells at 18 ℃ for 24 h. Under these conditions, the expression level of the phycobiliproteins was 149.5 mg/L. The optimal conditions for the induction of recombinant phycobiliproteins with lactose were adding 1.0 g/L of lactose and 5 mmol/L of 5-ALA to the bacterial culture at an OD600 of 1.2, followed by incubation at 18 ℃ for 26 h. The expression level in this case was 120.9 mg/L. The recombinant phycobiliproteins exhibited maximum absorbance at 550 nm and maximum emission at 562 nm. Compared to recombinant proteins obtained upon induction by IPTG, the recombinant proteins obtained upon induction by lactose had a higher absorbance at 550 nm, higher fluorescence emission at 562 nm, and higher hydroxyl radical and superoxide anion-scavenging activities.